Original Article
Rapid Frozen Section Diagnosis of Pituitary Tumors Hanne-Ooris Lang, Wolfgang Saeger, Dieter K. L0decke, and Dieter M011er
i~i !
Abstract
From 751 patients with suspected pituitary tumor, 2,354 frozen sections were prepared for histological identification of the tissue and determination of the resection margins after selective adenoma removal. The accuracy of the method was determined by comparison of the frozen section diagnoses with the diagnoses of permanent sections of the same tissue. The overall accuracy was 83. I%. The reasons for incorrect diagnoses in frozen section were analyzed retrospectively. Prevailing causes were spurious lesions resulting from problems in processing the extremely small specimens, and regressive transformations of the tissue leading to alterations of the tissue structure. Despite the rather low accuracy rate, we would consider frozen section diagnosis of pituitary tumors as a valuable aid for the surgeon. Other morphological methods are not as accurate in the determination of the resection margins in selective microsurgery. If applied by an experienced team, the method can be recommended. Endocr Pathol 1 : 1 1 6 - 1 2 2 , 1990,
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Departmentof Pathology of the Marienkrankenhaus (H-DL, WS) and Neurosurgical Department of the Universityof Hamburg (DKL, DM), Hamburg, F.R,G. Address correspondenceto: Dr. W. Saeger, Department of Pathologyof the Marienkrankenhaus,Alfredstrasse9, 2000 Hamburg 76, F.R.G.
During the past 20 years, selective transsphenoidal microsurgery has become the most common surgical treatment of pituitary adenomas (9,10). The main problem for the surgeon is determination of the resection margins after removal of the tumor, as pituitary adenomas do not have a fibrous capsule and small complexes of tumor cells often spread into grossly normal-looking tissue (1,11,20). Histological diagnosis provides help for the surgeon in this problem. There are only a few studies dealing with frozen section diagnosis of pituitary pathological processes: Adelman and Post (1) find the accuracy of this method to be approximately 90%. McKeever and colleagues (19) report accuracy rates of 91.4% and 94.3%. These studies reveal that frozen section diagnosis of pituitary abnormalities is not as reliable as in most other domains where this method is used. In breast pathological processes, accuracy rates of frozen sections are between 98.3% (8) and 99.7% (2). Studies dealing with frozen sections from several sorts of tissue
mention accuracy rates between 97.3% (6) and 99.3% (7). With frozen sections of the pituitary there are two basic problems: First, the specimens are extremely small (most less than 3 mm in diameter). This is a serious obstacle to technical quality in the preparations (17,18,23). Second, pituitary adenomas show only a few histological characteristics to distinguish them from normal pituitary tissue. These are a high cellularity and the uniformity of the cells (3,4,11,23). Some authors describe less distinct features such as uniform round nuclei of the cells and vcell-defined cytoplasmic borders (3) or a subtle perpendicular orientation of the cells to the septate fibrovascular stroma (4). In addition, the tissue of the pituitary anterior lobe can be altered by compression through the adjacent tumor (11,16). This study aims to determine the accuracy of frozen section diagnosis of pituitary tumors in 2,354 specimens. Specimens with incorrect diagnosis in frozen section are
Rapid Diagnosis of Pituitary Tumors
histologically reexamined to identify the reasons for misdiagnosis.
Material and Methods From 1973 to 1987, 2,354 specimens for rapid frozen section were removed from the pituitary gland of 751 patients with suspected pituitary tumor. The number of specimens per patient varied from 1 to 14. Frozen section diagnosis was performed for two reasons: (1) to identify suspected tumor tissue histologically, in particular to differentiate between adenoma, other tumor, hyperplasia of adrenocorticotropic hormone (ACTH) cells, and nontumorous tissue, and (2) to determine the adequacy of resection margins of a tumor. During surgery, specimens for frozen section were taken from the center of the suspected mass and from the resection margins after selective removal of the tumor. The size of the specimens ranged from 1 mm up to 3 mm. Additionally, specimens of the suspected tumor were taken from each patient to perform direct paraffin sections and immunohistological examinations covering all six pituitary hormones. In approximately half of the cases, further material was removed for epon-embedding and examination by electron microscopy. However, the results of these investigations are not considered in this study. After transport to the laboratory, the unfixed material was frozen with liquid nitrogen and then cut in the cryostat at -20~ (SLEE Kryostat HR, Mainz, F.R.G.). The thickness of the sections ranged from 6 to 8 lim. The sections were attached directly to warm slides, air-dried, and stained with standard rapid hematoxylin and eosin stain (5). The remaining frozen material was thawed, fixed, and then embedded into paraffin. Preparations of this material were stained both with hematoxylin and eosin and with periodic acid-Schiff (PAS)o The frozen sections were examined independently by one to three experienced investigators (one pathologist, one neuropathologist, and one neurosurgeon). These diagnoses were compared to the final diagnoses of the same specimen embedded in paraffin. As relevant diagnostic errors, we considered those discrepancies in diagnosis
1 17
concerning the differentiation between tumorous and nontumorous tissue (false-positive or false-negative diagnoses). Uncertain diagnoses were not regarded as diagnostic errors but as a limitation of the technique (13). We reexamined histologically frozen sections with wrong diagnoses and those frozen sections with uncertain diagnoses, which turned out to be wrong. We included only errors resulting from morphological misinterpretation. The preparations were reviewed for the following features: (1) presence of distinct types of tissue, (2) characteristics of architecture, (3) presence of artifacts, and (4) evidence of general technical shortcomings. Each frozen section was correlated to a maximum of three characteristics that retrospectively were regarded as distinct marks for diagnostic error.
Results Accuracy of Frozen Section Diagnosis of Pituitary Abnormalities In 2,354 frozen sections, there were 149 (6.3%) false-positive diagnoses, 242 (10.3%) false-negative diagnoses, and 8 (0.3%) diagnoses with wrong interpretation of the type of tumor, resulting in an overall accuracy of 83.1%. O f these diagnostic errors, 311 (77.9%) were caused by misinterpretation of the morphological structures in the frozen sections. Retrospectively, 88 (22.1%) of the errors resulted from an unequal dissemination of tumorous and nontumorous tissue in frozen section and paraffin section. There were 342 frozen sections with uncertain diagnoses, of which 143 (41.8%) turned out to be falsely positive, 24 (7.0%) turned out to be falsely negative, and 6 (1.8%) were uncertain concerning the type of tumor. Table 1 shows the accuracy of the frozen section diagnoses in relation to the clinical case diagnoses. Table 2 shows the accuracy in relation to the final histological case diagnoses. There are no significant differences in the results for diagnoses with high numbers of frozen sections such as the clinical diagnoses of Cushing's disease, acromegaly, prolactinoma, and inactive tumor, and the histological diagnoses of pituitary adenomas, hyperplasia of A C T H cells, and nontumorous tissue. In those cases, diagnostic accura-
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Table 1, The accuracy of frozen section diagnosis: comparison between the diagnoses in l~rozen sections of tumor and paratumorous tissue and the histological diagnoses in paraffin sections in relation to the clinical diagnoses
Clinical Diagnosis
Number of Cases
Cushing's disease Acromegaly Prolactinoma TSH-producing adenoma Inactive tumor Craniopharyngioma Other Total
Number of Frozen Sections
203 248 112 2 139 28 19
724 864 324 12 304 88 38
751
2,354
False Positive Diagnosis 66 (9.1%) 43 (5.0%) 5 (1.5%) -12 (3.9%) 20 (22.7%) 3 (7.9%) 149 (6.3%)
False Negative Diagnosis
Wrong Type of Tumor
Uncertain Diagnosis
Accuracy (%)
40 (5.5%) 105 (12.2%) 44 (13.6%) 4 (33.3%) 38 (12.5%) 6 (6.8%) 5 (13.2%)
-2 (0.2%) --5 (1.6%) -1 (2.6%)
146 (20.2%) 110 (12.7%) 33 (10.2%) 1 (8.3%) 34 (11.2%) 15 (17.0%) 3 (7.9%)
85.4 82.6 84.9 66.7 82.0 70.5 76.3
242 (10.3%)
8 (0.3%)
342 (14.5%)
83.1
TSH = thyroid-stimulating hormone.
Seven frozen sections did not reveal any histological features explaining the incorrect diagnoses.
cies are between 81.9% and 88.5%. Craniopharyngiomas have lower accuracy rates o f 70.5% (clinical diagnosis) and 74.0% (histological diagnosis). T h e remaining case diagnoses have more divergent accuracy rates, but the small n u m b e r o f frozen sections in these categories does not allow any conclusions to be drawn.
Special Types of Tissue. Extensive connective tissue o f focal or irregular nature is the most frequent histological attribute o f m i s d i agnosed frozen sections (41.8%). It represents a reaction of the tissue (in particular o f t u m o r tissue) to cell necroses and leads to fibrotic transformation, so that the original architecture is unrecognizable. An interstitial sclerosis o f the anterior lobe o f the pituitary as a consequence o f chronic compression does
Histological Reexamination of Misdiagnosed Frozen Sections
We reexamined 471 frozen section preparations. T h e results are summarized in Table 3.
Table 2.
The accuracy of frozen section diagnosis: comparison between the diagnoses in frozen sections of tumor and patatumorous tissue and the histological diagnoses in paraffin sections in relation to the final histological diagnoses (case diagnoses)
Final Histological Diagnosis Pituitary adenoma Craniopharyngioma Meningioma Hyperplasia of ACTH cells No tumor or hyperplasia Other Total
Number of Cases
Number of Frozen Sections
606 25 4
1,949 81 7
13
False Negative Diagnosis
Wrong Type of Tumor
97 (5.0%) 16 (19.8%) --
235 (12.1%) 5 (6.2%)
6 (0.3%)
61
7 (11.5%)
m
93 10
232 24
28 (12.1%) 1 (4.2%)
751
2,354
ACTH = adrenocortcotropic hormone.
False Positive Diagnosis
149 (6.3%)
Uncertain Diagnosis
Accuracy
270 (13.9%) 16 (19.8%) 3 (42.9%)
82.6 74.0 100.0
11 (18.0%)
88.5 87.9 79.2 83.1
2 (8.3%)
2 (83%)
40 (17.2%) 2 (8.3%)
242 (t03%)
8 (0.3%)
342 (14.5%)
(%)
Rapid Diagnosis of Pituitary Tumors
9
Table 3,
7
9
9
~9
:9
9
,
I 19
,
Histological characteristics of misdiagnosed frozen sections
Diagnosis
False Positive Diagnosis N = 259
False Negative Diagnosis N = 212
N = 47t
Total
Special types of tissue Septation by connective tissue and fibroses Interstitial sclerosis of the anterior pituitary Granulation tissue Blood or fibrin
77 (29.7%)
120 (56,6%)
197 (41.8%)
7 (2.7%) 46 (17.8%) 19 (7.3%)
17 (8,0%) 15 (7.1%) 18 (8.5%)
24 (5.1%) 6t (13.0%) 37 (7.9%)
Characteristics of architecture Inhomogeneous structure Pretended alveolar pattern Necrobioses and necroses
25 (9.6%) -6 (2.3%)
17 (8.0%) 5 (2.4%) 18 (8.5%)
42 (8.9%) 5 (1.1%) 24 (5.1%)
Spurious lesions Overlapping tissue in center of the frozen section Overlapping tissue at margin of the frozen section Ruptures or clefts of tissue Contusion of tissue Artificial formation of cell complexes Artifacts in center of the frozen section Artifacts at margin of the frozen section General technical shortcomings Frozen section too thick Frozen section too small Other
73 (28.2%)
60 (28.3%)
133 (28,2%)
6 (2.3%) 56 (21.6%) 10 (3.9%)
4 (l.9%) 49 (23.1%) 6 (2.8%)
10 (2.1%) 105 (22,3%) 16 (3.4%)
50 (19.3%)
25 (1t.8%)
75 (15.9%)
43 (16.6%)
67 (31.6%)
110 (23.4%)
15 (5.8%)
13 (6.1%)
28 (5.9%)
13 (5.0%) 19 (7.3%) 75 (29.0%)
9 (4.2%) 5 (2.4%) 49 (23.1%)
22 (4.7%) 24 (5.1%) 124 (26.3%)
not lead to substantial destruction of tissue architecture. It is found in 5,1% o f frozen sections as a reason for wrong diagnosis. The coincidence of tumorous and nontumorous pituitary tissue with regressive transformations and an extensive presence o f fibroses (Fig. 1A and B) represents a special reason for misdiagnosis. Granulation tissue may be responsible for errors in diagnosis in 13.0% of the misdiagnosed frozen sections. One-third of these sections are cases ofcraniopharyngioma, and so they cannot truly be classified as
misdiagnoses since craniopharyngiomas always tend toward extensive regressive transformation and finally create masses o f granulation tissue. Such findings may be considered sufficient evidence for craniopharyngioma. Original epithelial cells as final histological proof for the tumor can be found only in isolated areas of the specimens. Characteristics of Architecture. An inhomogeneous structure of the tissue is caused by nonspecific alterations of the cell architecture resulting in a tumor of uncommon structure or seeming pleomorphism of the cells. This is found in 8.1% o f the misdiagnosed frozen sections. A special form of this characteristic is an apparent alveolar pattern of tumorous tissue (1.1%) that imitates normal tissue of the anterior lobe.
Artifacts. All of these characteristics represent effects o f the technical processing o f the tissue. The most sensitive steps are the freezing process and the cutting in the cryostat. Most of the specimens show several signs of technical shortcomings so that it is impossible to give a specific reason for a specific characteristic. Overlapping tissue in the center o f the frozen sections (28.2%) and ruptures and clefts o f tissue (22.3%) make it difficult to interpret the tissue structure, especially if different types of tissues are situated close together. Artifacts are found in the center of the preparations in 23.4% and at the margins in 5.9%. In 15.9% of the misdiagnosed frozen sections, an artificial formation o f cell complexes imitates solid cell complexes oftumoro General Technical Shortcomings. General technical shortcomings, such as too thick or extremely small sections with only isolated groups o f cells, occur in 36.1% o f all misdiagnosed frozen sections. Specific Diagnostic Problems. Specific diagnostic problems concern the type o f tumor when it shows an uncommon structure in frozen section. Meningiomas especially tend to imitate pituitary adenomas by apparently having an epithelial character. Only the paraffin section reveals that the tumor is comprised o f fibers and whorled formations (Fig. 2A and B).
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noma, and found an accuracy of 90%. In contrast to our study, they used an orange G-hematoxylin method to improve the staining of different cell types. McKeever and co-workers (19) investigated 35 frozen sections from biopsy and autopsy material from the pituitary and applied hematoxylin and eosin stain as wetl as a new Ricinus communis agglutinin 120 (RCA 120) stain. With the conventional technique, they achieved a diagnostic accuracy of 91.4% compared to 94.3% with the RCA 120 stain. The studies mentioned here reflect accuracies that are approximately 10% higher than in our study. The different result may be attributed to the fact that we included not only the easily defined tumor tissue but also the border areas of the gland. Moreover, the low number of frozen sections allows only limited comparison to our study. p.--?r
-),
;: 9 Reasons for Diagnostic Errors
Specimens" Small Dimension.
Specimens of
the pituitary measuring between 0.5 and 1.5 mm in diameter are very difficult to process without producing serious technical deficiencies (16-18,23). In our study, we attempted to prepare all specimens for frozen section, even if they were extremely small. Spurious lesions were found in approximately 25% of our sections and represent the result of the problems in processing such small specimens. Actually, 2.6% of the frozen sections did not contain enough material to allow any interpretation at all.
B
Figure 1.
(A) Frozen section of an oncocytic adenoma {adjudged as anterior pituitary tissue with regressive transformations by two investigators). Epithelial tumor with extensece fibroses in the form of stripes or reticular structure, tn the upper part of the preparation, there are areas of tissue that might represent tissue of the anterior lobe of the pituitary with a trabecular transformation of its structure, a broad stroma, and fibroses (H&E, x90). (B) Paraffin section of the sa~ne oncocytic adenom& Tumor with necrotic tissue and numerous fibroses (PAS, x225),
Discussion Accuracy o f Frozen Section Diagnosis of Pituftary A b n o r m a l i t i e s
Our study of 2,354 frozen sections of the pituitary from 751 patients with suspected pituitary tumor reveals an overall accuracy of 83.1%. Adelman and Post (1) examined 200 frozen sections, taken from 30 patients with microadenomas after resection of the ade-
Difficulty In Distinguishing Normal Pituitary from Adenoma, The histological characteris-
tics causing misdiagnosis in frozen section mostly reveal regressive transformations of the tissue leading to proliferation of connective tissue and to necroses. They are specific characteristics of some tumors as well as general reasons for misinterpretation that are mentioned also in other domains where the method is used (22). A focal, septate fibrous stroma occurs especially in pituitary adenomas of solid architecture (20). Masses of granulation tissue are a specific result of regressive transformation in craniopharyngiomas (20,21). Signs of an inhomogeneous structure are also reported by Burger 0), who mentions that the freezing process tends to angulate
Rapid Diagnosis of Pituitary Tumors
1 21
nuclei to produce pleomorphism. As a consequence, craniopharyngioma, metastatic carcinoma, and other sellar tumors must be considered in the dif[~rential diagnosis. Sometimes, frozen sections o f adenomas show cholesterol crystals and foreign-body giant cells, making craniopharyngioma a consideration (23). P s a m m o m a bodies or whorl formations in meningiomas may be mistaken for calcopherites o f pituitary adenomas.
Figure 2,
(A) Frozen section of a meningioma (with two uncertain and one definite diagnosis of a pituitary adenoma in frozen section). Epithelial-looking tumor with monomorphic cells of intermediate or large size (H&E, x90). (B) Paraffin section of the same meningioma. Tumor with pleomorphic nuclei, an extracted-looking cytoplasm, and netlike or whorled formations of fibers, There is a rather high proportion of capillaries and, in some parts, a distinct pericapillary fibrovascutar stroma. No anterior pituitary tissue can be seen, Retrospectively, a definite diagnosis is possible only in context with the paraffin section (PAS, x225).
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Other Diagnostic Methods Because o f the rather low accuracy rates o f frozen sections o f the pituitary, some authors introduced stains different from hematoxylin and eosin (1,24). The reticulum stain developed by Velasco and colleagues (24) deserves special attention because it demonstrates very clearly the differences in the reticulin fiber network between adenoma and normal pituitary. However, the authors do not give any information about accuracy rams using this technique. We agree with Kovacs and H o r vath (1l) that this staining procedure should be reserved for some rare cases as an additional method. In contrast to frozen sections, smears and touch preparations can be performed more quickly and do not lead to almrations o f the tissue structure through a freezing process (3,12,17,18). Very little material is needed, so that tissue can be spared for permanent sections. However, in contrast to frozen sections, touch preparations do not reveal details o f tissue architecture (11). Since specimens for identification o f suspected tumor usually show only one type o f cell, touch preparations are sufficient for this diagnosis. Nonetheless, from our experience, touch preparations are not useful for our main indication for frozen section diagnosis-namely, to determine the resection margins. Additional information about the tissue structure is necessary because the different cell types represented in these specimens lead to diagnostic problems in touch preparations. Some new mchniques for intraoperafive h o r m o n e measurement have been developed by Liidecke (14,15), such as intraoperative measurements o f A C T H to localize microadenomas in Cushing's disease and intraoperative measurements o f growth h o r m o n e decrease in patients with acromegaly to assure the complete removal o f an adenoma. H o w ever, both techniques are reserved for special centers with an attached neuroendocrinological laboratory.
Value of Frozen Sections in Pituitary Surgery
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Today, selective removal o f adenomas is the ideal goat in the surgical treatment o f pituitary tumors because tissue for pituitary function is preserved. This operative technique can be performed only by very experienced neurosurgeons. Even so, grossly distinguishing ade-
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noma or other tumor from normal pituitary and determining the exact resection margins may be difficult for the surgeon. As selective operative techniques do not allow the surgeon to remove specimens o f such a size that technical deficiences in frozen section diagnosis can be avoided, this method will always have limited accuracy. From our experience, the rather low accuracy rate o f frozen section diagnosis o f pituitary abnormalities must be tolerated for there are no more reliable methods. Frozen section diagnosis can be recommended if it is performed by an experienced team of neurosurgeons and pathologists who are conscious o f the problems o f both the technique and interpretation. A prospective study will show whether the results can be further improved.
References 1. Adelman LS, Post KD. Intra-operative frozen section technique for pituitary adenomas. Am J Surg Pathol 3:173-175, 1979. 2. Agnantis NJ, Apostotikas N, Christodoulou I, Petrakis C, Garas J. The reliability of frozensection diagnosis in various breast lesions: A study based on 345t biopsies. Recent Results Cancer Res 90:205-210, 1984. 3. Burger PC. Use of cytological preparations in the frozen section diagnosis of central nervous system neoplasia. AmJ Surg Pathol 9:344-354, 1985. 4. Burger PC, Vogel FS. Frozen section interpretation in surgical neuropathology: I. Intracranial lesions. Am J Surg Pathol 1:342-347, 1977. 5. Culling CFA. Handbook of Histopathological and Histochemical Techniques (3rd ed.). London: Butterworth, 1974. 6. Dalai BI, MalikAK, Datta BN. Frozen section diagnosis--a review of 1051 cases. Indian J Cancer 16:59-65, 1979. 7. Eisner B. La biopsia por congelaci6n: Su valor asistencial y e n la educaci6n medica del patologo. Prenat Med Argent 55:1741-1749, 1968. 8. Fessia L, Ghiringhello B, Arisio R, Botta G, Aimone V. Accuracy of frozen section diagnosis in breast cancer detection. A re,Aewof 4436 biopsies and comparison with cytodiagnosis. Pathol Res Pract 179:61-66, 1984. 9. Hardy J. Transsphenoidal microsurgery of the normal and pathological pituitary. Clin Neurosurg 16:185, 1969. 10. HardyJ. Transsphenoidal Microsurgical Treatment of Pituitary Tumors. In: Linfoot JA ed.
Recent Advances in the Diagnosis and Treatment of Pituitary Tumors. New York: Raven Press, 1979. Pp. 375-388. 11. Kovacs K, Horvath E. Tumors of the Pituitary Gland. Washington, DC: Armed Forces Institute of Pathology, 1986. Pp. 51-56. 12. Landolt AM, Krayenbtihl H. A modified cytological technique for rapid differentiation of pituitary adenomas. J Neurosurg 37:289293, 1972. 13. Lang H-D. Untersuchung zur Anwendung der intraoperativen Schnellschnittdiagnostik bei Hypophysentumoren. Inaugural-dissertation, University of Hamburg, 1989. 14. Lfidecke DK. Longterm surgical results in acromegaly. Does the immediate postoperative growth hormone level predict the outcome? Adv Biosciences 69:243-251, 1988. 15. Liidecke DIC Intraoperative measurement of adrenocorticotropic hormone in peripituitary blood in Cushing's disease. Neurosurgery 24:201-205, 1989. 16. Martinez AJ, Lee A, Moossy J, Maroon JC. Pituitary adenomas: Clinicopathological and immunohistochemical study. Ann Neurol 7:24-36, 1980. 17. Martinez AJ, MoossyJ. Frozen sections, touch preparations and other technical procedures in the diagnosis of pituitary adenomas. Presented at a meeting of the Pituitary Pathology Group, Montreal, Canada, September 30, 1981. 18. Martinez AJ, Moossy J. Cytological diagnosis of pituitary adenomas. J Neuropathol Exp Neuro142:307, 1983. 19. McKeever PE, Laverson S, Oldfield EH, Smith BH, Gadille D, Chandler WF. Stromal and nuclear markers for rapid identification of pituitary adenomas at biopsy. Arch Pathol Lab Med 109:509-514,1985. 20. Saeger W. Hypophyse. In: Doerr W, Seifert G (Hrsg.). Spezielle pathologische Anatomie, Band 14/1, Pathologie der endokrinen Organe. Berlin: Springer-Verlag, 1981. Pp. 1226. 21. Saeger W, K16ppel G, Riede UN. Endokrines System. In: Riede U-N, Wehner H (Hrsg.). Allgemeine und spezielle Pathologie. Stuttgart: Thieme, 1986. Pp. 819 ft. 22. Saltzstein SL, Nahum AM. Frozen section diagnosis: Accuracy and errors; uses and abuses. Laryngoscope 83:1128-1143, 1973. 23, Takei 3(. Pathology of Pituitary Tumors and Value of Frozen Section Diagnosis. In: Tindall GT, Collins WF (eds.). Clinical Management of Pituitary Disorders. New York: Raven Press, 1979. Pp. 93-112. 24. Velasco ME, Sindely SD, Roessmann U. Reticulurn stain for frozen-section diagnosis of pituitary adenomas. Technical note. J Neurosurg 46:548-550, 1977.
